The present invention is concerned with a method for the preparation of an infectious IBDV mutant capable of replication in CEF cell culture, a genetically engineered IBDV mutant as well as with a vaccine comprising such a IBDV mutant.
Infectious bursal disease virus (IBDV) is a member of the Birnaviridae family. Viruses in this family have a very similar genomic organisation and a similar replication cycle. The genomes of these viruses consist of 2 segments (A and B) of double-stranded (ds) RNA. The larger segment A encodes a polyprotein which is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4. VP2 and VP3 are the major structural proteins of the virion. VP2 is the major host-protective immunogen of bimaviruses, and contains the antigenic regions responsible for the induction of neutralising antibodies. The VP4 protein appears to be a virus-coded protease that is involved in the processing of a precursor polyprotein of the VP2, VP3 and VP4 proteins. The larger segment A possesses also a second open reading frame (ORF), preceding and partially overlapping the polyprotein gene. This second open reading frame encodes a protein VP5 of unknown function that is present in IBDV infected cells. The smaller segment B encodes VP1, a 90 kDa multifunctional protein with polymerase and capping enzyme activities.
For IBDV, two serotypes exist, serotype 1 and 2. The two serotypes may be differentiated by virus neutralisation (VN) tests. Furthermore, subtypes of serotype 1 have been isolated. These so-called xe2x80x9cvariantxe2x80x9d viruses of serotype 1 can be identified by cross-neutralisation tests, a panel of monoclonal antibodies or RT-PCR. These subtypes of serotype 1 of IBDV have also been described in literature, for example: classical, variant-E, GLS, RS593 and DS326 strains (Van Loon, et al. Proceedings of the International symposium on infectious bursal disease and chicken infectious anaemia, Rauischholzhausen, Germany, 179-187, 1994).
Infectious Bursal disease (IBD), also called Gumboro disease, is an acute, highly-contagious viral infection in chickens that has lymphoid tissue as its primary target with a selective tropism for cells of the bursa of Fabricius. The morbidity rate in susceptible flocks is high, with rapid weight loss and moderate mortality rates. Chicks that recover from the disease may have immune deficiencies because of the destruction of the bursa of Fabricius which is essential to the defence mechanism of the chicken. The IBD-virus causes severe immunosuppression in chickens younger than 3 weeks of age and induces bursal lesions in chicks up to 3 months old.
For many years the disease could be prevented by inducing high levels of antibodies in breeder flocks by the application of an inactivated vaccine, to chickens that had been primed with attenuated live IBDV vaccine. This has kept economic losses caused by IBD to a minimum. Maternal antibodies in chickens derived from vaccinated breeders prevents early infection with IBDV and diminishes problems associated with immunosuppression. In addition, attenuated live vaccines have also been used successfully in commercial chicken flocks after maternal antibodies had declined.
Recently, very virulent strains of IBDV have caused outbreaks of disease with high mortality in Europe. The current vaccination programs failed to protect chicks sufficiently. Vaccination failures were mainly due to the inability of live vaccines to infect the birds before challenge with virulent field virus.
Therefore, a constant need exists to improve existing vaccines and to develop new types of vaccines. For the development of live vaccines IBD viruses in attenuated form are required. Conventionally, this can be achieved by serial passaging of IBDV field isolates on an appropriate substrate. For the development of inactivated IBDV vaccines, an appropriate substrate is necessary for the generation of high amounts of IBDV antigen mass resulting from the propagation of IBD viruses on the substrate.
It is known that field IBDVs can readily be propagated in vivo in the bursa of infected birds or in embryonated eggs. However, although, the successful adaptation an propagation of some IBDV strains to in vitro cell culture of chicken embryo origin has been reported, it is generally acknowledged that most IBDV strains isolated from infected bursa in the field, in particular the so-called virulent- or very virulent IBDV strains cannot be adapted to cells of chicken embryo origin, such as chicken embryo fibroblasts (CEF) or cells from other organs such as the kidney and liver (Brown et al., J. Gen. Virology 75, 675-680, 1994; van Loon, et al., 1994, supra).
The drawbacks of the in vivo culture substrates are obvious. Such culture methods are animal unfriendly, need a lot of animals, are time consuming and cannot be carried out under standardised and stringent conditions.
In addition, the limited number of IBDV strains which are not refractory to adaptation to in vitro cell culture substrates, suffer from the disadvantage that as a result of the serial passaging process leading to the adaptation of the IBDV strains, random mutations are introduced in the genome of the virus in an uncontrolled manner. Such mutations may influence properties of the virus other than that associated with the adaptation of the virus to the cell culture, e.g. properties related to the immunogenicity of the virus. Such additional, random mutations are not desired. The adaptation of the IBDVs by passaging of the virus in vitro in CEF cell cultures has been associated with attenuation of the virulence as demonstrated by a reduction of the virus"" ability to induce lesions in the bursa of the infected bird. Yamaguchi et al. (Virology 223, 219-223, 1996) investigated the molecular basis for the virulence of IBD viruses and the attenuation of these viruses as a result of the adaptation of bursa IBDVs to CEF cell culture. It was concluded that from the studies carried out by Yamaguchi et al. the precise mutations involved in attenuation of the wild-type IBDV could not be identified. It was suggested that the amino acid residues at position 279 (Asp/Asn) and 284 (Ala/Thr) of the polyprotein encoded by the long open reading frame of the segment A are important for virulence or propagation of the IBDV in CEF cells.
The latter was confirmed by Lim, B-L (Proceedings of the 4th Asia Pacific Poultry Health Conference, Nov. 22-26, 1998, Melbourne, Australia, Abst. 79). It is disclosed therein that substitution of the amino acid residues 279 (Aspxe2x86x92Asn) and 284 (Alaxe2x86x92Thr) in the VP2 protein of an IBDV results in a IBDV mutant which can be propagated in CEF cell culture. However, the prior art does not teach an alternative of type and minimal number of amino acid mutations which are required and sufficient to allow the adaptation of bursa IBDV to CEF cell culture.
It is an object of this invention to provide a generally applicable method for adaptation of IBDV isolates which only grow in vivo in the bursa of infected birds to growth in cell culture.
It is a further object of the present invention to provide a method for preparing attenuated IBDV mutants by introducing mutations in the IBDV genome in a controlled manner.
Moreover, it is an object of the present invention to provide a genetically engineered IBDV mutant comprising the appropriate amino acid residues which allow the mutant to grow in cell culture.
It has been found that this object has been met by a method for the preparation of an infectious IBDV mutant capable of replication in CEF cell culture comprising the steps of, (i) separately preparing a DNA construct comprising cDNA of genome segments
A and B of an IBDV not capable of replication in CEF cell culture,
(ii) introducing a mutation in:
a one or more codons for amino acid residues 253 and 284 of the VP2 gene of Variant-E or Classical IBDV strains, or in
b a codon for amino acid residue 284 of the VP2 gene of a GLS IBDV strain, on the cDNA comprising the segment A, such that the codons for amino acid residues 253 and 284 of the mutated VP2 gene encode a histidine and threonine residue, respectively, in the case of a Variant-E or Classical IBDV strain, or such that the codon for amino acid residue 284 of the mutated VP2 gene encodes a threonine residue in the case of a GLS IBDV strain,
(iii) allowing RNA transcripts of the cDNA comprising the segment A and the segment B to initiate replication of the IBDV mutant in host cells in a culture medium, and
(iv) isolating the IBDV mutant from the culture.
The present invention for the first time identifies which amino acid residues are required and sufficient to allow an IBDV to replicate in CEF cell culture. Whereas most IBDV bursa-isolates comprise the amino acid residues 253 (Gln), 284 (Ala) and 330 (Ser) of the VP2 protein, Variant-E or Classical IBDV mutants whose codons at position 253 and 284 have been changed such that they now encode the amino acid residues 253 (His) and 284 (Thr) are able to grow in CEF cell culture.
For GLS IBDV mutants it has been found that it is sufficient to change the codon at position 284 such that it now encodes the amino acid residue 284 (Thr).
It has also been found that the amino acid position 330 is not critical for the replication of the Classical or Variant-E IBDV mutant in CEF. However, serine, arginine or lysine residues are most favoured at that position in the present invention.
Additionally, it has been found that for GLS IBDV amino acid position 253 is not critical for replication, and usually is a glutamine residue.
Hence, in a preferred method an IBDV mutant is prepared which comprises any of these three amino acid residues, in particular 330 (Arg), at this position in the VP2 protein. For GLS IBDV mutants the preferred amino acid residue at position 253 is a glutamine.
It has been found that in the genome of a (chimeric) Variant-E IBDV which was able to replicate only in the bursa of infected chickens (D78/Variant-E), two changes are necessary to adapt IBDV isolates to CEF cell culture. These positions involve amino acid residue 253 and 284. Amino acid residues histidine and threonine at these positions, respectively, allow the IBDV mutant to replicate in CEF cell culture. (Table 1 and Example 1). Furthermore, it has been found that the IBDV mutants adapted to CEF cell culture according to the method of the invention are also attenuated (Example 2).
To further proof that the adaptation from bursa to CEF cells for Classical IBDV strains is also determined by the two amino acids, the codons of IBDV strain D78 which is able to replicate in CEF cell culture were changed at positions 253, 284 and 330. The results are shown in Table 2A.
Classical xe2x80x9cvery virulentxe2x80x9d (VV) European isolate UK661 (Brown et al., J. Gen. Virology 75, 675-680, 1994; Brown and Skinner, Virus Res. 40, 1-15, 1996) cannot be propagated in vitro, and therefore has to be propagated in vivo in chickens. Chickens have to be infected with the VV strain and a few days post-infection surviving birds are killed and the bursa is removed. The virus can then be extracted from bursal homogenate for further use. The experiments underlying the present invention demonstrated that the amino acid changes at positions 253 and 284 as defined above allow the VV strain UK661 to growth in cell culture. The results of the mutagenesis and transfection experiments with this Classical IBDV strain are summarised in Table 2B.
These data further proof that the amino acid changes at positions 253 and 284 are sufficient to allow Classical bursa IBDV strains to growth in cell culture. All other mutations result in mutants which either do not replicate in cell culture or replicate very poorly (see also Lim et al., J. Virol. 73, 2854-62, 1999).
Additionally, it was determined that in GLS IBDV the exchange of a single amino acid residue at position 284 was sufficient to allow a bursa adapted IBDV to replicate on CEF cells (Table 3).
Therefore, the method according to the invention allows the adaptation of IBDV bursa-isolates to growth in cell culture by means of recombinant DNA techniques. The advantage of the present method is that as a result of the adaptation process only mutations are introduced in the genome of the IBDV in one or more of the codons at position 253 and 284. The numbers indicate the amino acid and codon positions of the polyprotein and large open reading frame on segment A of the IBDV genome, respectively (Mundt and Mxc3xcller, J. Gen. Virol. 77, 437-443, 1995; NCBI accession number X 84034).
Most but not all IBDV strains which fail to grow on CEF cell culture contain the codons 253 (Gln), 284 (Ala) and 330 (Ser) in the VP2 gene. Some Variant-E or Classical IBDV strains which fail to grow on CEF cell culture may already have one of the required codons 253 (His) and 284 (Thr). Therefore, the method according to the invention comprises the introduction of a mutation in one or two of the required codons mentioned above, such that the resulting IBDV mutant comprises the codons in the VP2 gene encoding the amino acid residues 253 (His) and 284 (Thr).
More preferably, the method of the invention is applied to an IBDV which is not capable of replication in CEF cells and which comprises the codons for amino acid residues 253 (Gln) and 284 (Ala), and even more preferably 330 (Ser). In the case of Classical and Variant-E strains, mutations are introduced in the two or three of the codons of the VP2 gene resulting in the codons 253 (His) and 284 (Thr), and optionally 330 (Arg). The new codons for the amino acids at these positions may be: for His (CAT or CAC), for Thr (ACT, ACC, ACA, ACG) and for Arg (CGT, CGC, CGA, CGG, AGA, AGG).
Even more preferably, the method of the invention is applied to an IBDV which is not capable of replication in CEF cells and which comprises the codons Gln 253 (CAA), Ala 284 (GCC) and optionally Ser 330 (AGT) or any combination thereof.
In particular, the method of the invention is applied to an IBDV which is not capable of replication in CEF cells and which comprises the codons 253 (CAA), 284 (GCC) and 330 (AGT).
The method for the preparation of an IBDV mutant according to the present invention comprises the recently established xe2x80x9creverse geneticsxe2x80x9d system for birnaviruses (Mundt and Vakharia, Proc. Natl. Acad. Sci. USA 93, 11131-11136, 1996 and WO 98/09646). This reverse genetics system opened the possibility to introduce mutations in the RNA genome of an IBD virus. The principle of the reverse genetics method according to the invention is that genomic RNA segments A and B are isolated from the virus, followed by reverse transcription of the RNAs into cDNA, after which the cDNAs are transcribed into RNA. The introduction of the required mutation(s) into the segment A (or B) of the virus takes place at the cDNA level. An important step in this reverse genetics system is to provide separate DNA constructs comprising a DNA vector molecule (e.g. a plasmid) and full length cDNA clones of the segments A or B of the IBDV. DNA constructs comprising the segment A or B cDNA, including the nucleotides of the 5xe2x80x2- and 3xe2x80x2-ends of both these segments can be generated according to the method described by Mundt and Vakharia (1996, supra). The subsequent step in the reverse genetics method is the transfection of suitable host cells with appropriate segment A and B genetic material such that in the transfected host cells RNA transcripts of cDNA segment A and B can initiate replication of the virus, resulting in infectious IBDV which can be isolated from the medium in which the host cells are cultured.
Several methods for the latter step of the reverse genetics system may be used. Preferably, the method according to the invention comprises the preparation of synthetic RNA transcripts from both the segment A and B cDNAs in vitro. In this case the DNA constructs comprise a RNA polymerase promoter operably linked to either of the segments. The promoter can be the promoter for the T7, SP6 or T3 polymerase, the T7 promoter being preferred. The synthetic transcripts of the A and B segment are isolated and used to transfect suitable host cells.
Alternatively, a method is provided in which a cell line is provided comprising host cells capable of expressing a RNA polymerase and which are transformed with a DNA construct comprising cDNA of segment B and a RNA polymerase promoter, such that RNA transcripts of segment B are constitutively expressed. After transfection of such cells with a synthetic RNA transcript of the cDNA comprising mutated segment A, the replication of the IBDV mutant is initiated in the host cells. In particular, host cells may be used which are able to express bacteriophage T7 DNA-dependent RNA polymerase, expressed for example cytoplasmically from recombinant vaccinia virus.
The desired mutations can be introduced into the VP2 gene by means of methods generally known in the art for this purpose. In particular, the mutation(s) are introduced by means of site-directed mutagenesis. Methods for introducing a mutation in the IBDV genome are described herein, but are also generally used in the art (Mundt and Vakharia, 1996, supra; Yao et al., J. Virology 72, 2647-2654, 1998; Mundt et al. European patent application no. 0887,412 and Current Protocols in Molecular Biology, eds.: F. M. Ausubel et al., Wiley N.Y., 1995 edition, pages 8.5.1.-8.5.9.)
The method according to the invention may be applied to all IBDV strains which are not capable of replication in CEF cell culture, and which are of the Classical, Variant-E, or GLS antigenic sub-types of IBDV.
Moreover, the method according to the invention may be applied to all IBDV strains which are not capable of replication in CEF cell culture, independent of the virulence of the strains, and includes very virulent strains (such as CS89 and UK661), virulent strains (such as F52/70 and STC) and vaccine strains (such as 228E and 2512). The IBDV mutants which are adapted to replication in cell culture derived from very virulent- and virulent strains will be less virulent and may be used as live vaccine strains. Alternatively, such IBDV mutants can be propagated conveniently in cell culture and formulated as inactivated vaccines.
The method according to the invention may also be advantageously applied to IBDV attenuated strains which are not capable of replication in CEF cell culture. The mutants derived from such attenuated viruses can be used in a cell culture system for vaccine production in stead of in an in vivo production system.
According to a further aspect, the present invention provides a method for the preparation of a xe2x80x9cchimericxe2x80x9d IBDV mutant capable of replication in CEF cell culture. The method comprises the additional step of introducing a mutation in a gene of the segment A, preferably the VP2 gene, of a first IBDV, as a result of which the protein expressed by that gene comprises an epitopic determinant of a second IBDV.
A chimeric IBDV is a virus which comprises as a genetic backbone the segment A or VP2 gene of a first antigenic sub-type, and additionally comprises the genetic information encoding an epitopic determinant of a second IBDV antigenic sub-type. In particular, such chimeric IBDVs express one or more additional epitopic determinants on the VP2 protein of the IBDV of the first antigenic sub-types. The advantage of such a chimeric IBDV is that can be used as a single immunogen which induces immunity against at least two antigenic sub-types of IBDV.
In particular, IBDV mutants are prepared which comprise the segment. A backbone or VP2 gene of Classical, GLS or Variant-E IBDV. cDNA clones containing the entire coding region of the segment A of the various IBDV strains can be prepared using standard cloning procedures and methods described in the prior art (Vakharia et al., Avian Diseases 36, 736-742, 1992; J. Gen. Virology 74, 1201-1206, 1993). The amino acid sequences and nucleotide sequences of the segment A of various IBDV strains are disclosed in the prior art (e.g. WO 95/26196 and Vakharia et al., Avian Diseases 36, 736-742, 1992).
Moreover, WO 95/26196 discloses the amino acid sequence of several epitopic determinants of the IBDV antigenic sub-types which are characteristic for each antigenic subtype In addition, WO 95/26196 discloses the antigenic characterisation of various IBDV strains by their reactivity with a panel of neutralising monoclonal antibodies. Important, epitopic determinants reactive with such neutralising Moabs are the B69 (classic sub-type), R63 and 67 (variant-E) and 57 (GLS) epitopic determinants. The region of the VP2 protein comprising the amino acid sequences for these epitopic determinants are described in Vakharia et al. (Virus Res. 31, 265-273, 1994)
Preferably, in the method according to the present invention a chimeric IBDV mutant capable of replication in CEF cell culture is prepared which comprises a classic segment A backbone and the nucleotide sequence encoding the variant-E epitopic determinant 67, or GLS epitopic determinant 57. Alternatively, the chimeric IBDV mutant comprises a GLS backbone and nucleotide sequences encoding the B69, R63 or 67 epitopic determinant.
In particular, the method according to the invention comprises the preparation of a chimeric IBDV strain (D78/Varaint-E) derived from strain D78 (commercially available from Intervet International B.V., the Netherlands) in which (i) the VP2 gene is replaced by the VP2 gene of a Variant-E strain, and (ii) the codons at positions 253, 284 and 330 are altered as defined-above (Example 1). Basically, the steps for the introduction of nucleotide sequences encoding the epitopic determinants in the backbone segment A of a first IBDV are in essence the same as those for the introduction of the mutations defined-above. This is most easily done by providing cDNA of the genome segments A and B and (i) replacing the coding sequence for the epitopic determinant of the first IBDV by that of the second IBDV, or (ii) altering a specific codon in the first IBDV by site-directed mutagenesis. Such methods are also described in WO 95/26196. Finally, RNA transcripts of these cDNA molecules are allowed to initiate replication in a transfected host cell to obtain infectious, chimeric IBDV.
In another embodiment of the invention a method is provided for the preparation of and IBDV mutant as defined above, wherein the resulting IBDV mutant also comprises other mutations which attenuate the virus. An example of such a mutation is a mutation in the VP5 gene of the segment A of the IBDV genome resulting in an IBDV mutant which is not able to express a native VP5 protein. The preparation of an IBDV VP5xe2x88x92 mutant is described in European patent application No. 887,412.
According to a further aspect, the present invention provides a genetically engineered, infectious IBDV mutant capable of replication in CEF cell culture, comprising codons 253 (His) and 284 (Thr), and optionally 330 (Arg) in the VP2 gene of Classical or Variant-E strains, or codon 284 (Thr) of a GLS strain.
Such IBDV mutants still comprise the genetic information of bursa IBDVs which are not capable of replication in CEF cell culture, with the exception of the new codons mentioned-above which have been introduced in a controlled manner by means of genetic engineering techniques.
In particular, a Variant-E IBDV mutant as defined-above is provided which does not have glycine and/or valine on positions 318 and 325, respectively. Genetically engineered Variant-E mutants having aspartic acid and/or methionine at these positions, respectively, are most preferred.
In a preferred embodiment, the genetically engineered IBDV mutant according to the invention is a chimeric IBDV mutant, in particular a chimeric IBDV mutant derived from strain D78, comprising the nucleotide sequence encoding the VP2 gene of a Variant-E strain and having the three new codons specified above.
The present invention provides the possibility to easily prepare IBDV vaccines from IBDV strains which were previously refractory to replication in vitro cell culture. An additional advantage of the present invention is that IBDVs can be (further) attenuated in a controlled manner by the method described-above. Such attenuated IBDV mutants may be used as the active components in live IBDV vaccines.
Therefore, another aspect of this invention is a vaccine for use in the protection of poultry against disease resulting from IBDV infection. The vaccine comprises a genetically engineered IBDV mutant as prepared above, together with a pharmaceutical acceptable carrier or diluent.
The IBDV mutant can be incorporated into the vaccine as live attenuated or inactivated virus.
A vaccine according to the invention can be prepared by conventional methods such as for example commonly used for the commercially available live- and inactivated IBDV vaccines. Briefly, a susceptible substrate is inoculated with an IBDV mutant according to the invention and propagated until the virus replicated to a desired infectious titre after which IBDV containing material is harvested.
Every substrate which is able to support the replication of IBDV mutants can be used to prepare the vaccine according to the present invention, including primary (avian) cell cultures, such as chicken embryo fibroblast cells (CEF) or chicken embryo liver cells (CEL), mammalian cell lines such as the VERO cell line or the BGM-70 cell line, or avian cell lines such as QT-35, QM-7 or LMH. Usually, after inoculation of the cells, the virus is propagated for 3-10 days, after which the cell culture supernatant is harvested, and if desired filtered or centrifuged in order to remove cell debris.
Alternatively, the IBDV mutant is propagated in embryonated chicken eggs. In particular, the substrate on which these IBDVs are propagated are SPF embryonated eggs. Embryonated eggs can be inoculated with, for example 0.2 ml IBDV mutant containing suspension or homogenate comprising at least 102 TCID50 per egg, and subsequently incubated at 37xc2x0 C. After about 2-5 days the IBD virus product can be harvested by collecting the embryo""s and/or the membranes and/or the allantoic fluid followed by appropriate homogenising of this material. The homogenate can be centrifuged thereafter for 10 min at 2500xc3x97g followed by filtering the supernatant through a filter (100 xcexcm).
The vaccine according to the invention containing the live virus can be prepared and marketed in the form of a suspension or in a lyophilised form and additionally contains a pharmaceutically acceptable carrier or diluent customary used for such compositions. Carriers include stabilisers, preservatives and buffers. Suitable stabilisers are, for example SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried milk serum, albumin or casein) or degradation products thereof. Suitable buffers are for example alkali metal phosphates. Suitable preservatives are thimerosal, merthiolate and gentamicin. Diluents include water, aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol).
If desired, the live vaccines according to the invention may contain an adjuvant. Examples of suitable compounds and compositions with adjuvant activity are the same as mentioned below.
Although administration by injection, e.g. intramuscular, subcutaneous of the live vaccine according to the present invention is possible, the vaccine is preferably administered by the inexpensive mass application techniques commonly used for IBDV vaccination. For IBDV vaccination these techniques include drinking water and spray vaccination.
Alternative methods for the administration of the live vaccine include in ovo, eye drop and beak dipping administration.
In another aspect of the present invention a vaccine is provided comprising the IBDV mutant in an inactivated form. The major advantage of an inactivated vaccine is the high levels of protective antibodies of long duration that can be achieved.
The aim of inactivation of the viruses harvested after the propagation step is to eliminate reproduction of the viruses. In general, this can be achieved by chemical or physical means. Chemical inactivation can be effected by treating the viruses with, for example, enzymes, formaldehyde, xcex2-propiolactone, ethylene-imine or a derivative thereof. If necessary, the inactivating compound is neutralised afterwards. Material inactivated with formaldehyde can, for example, be neutralised with thiosulphate. Physical inactivation can preferably be carried out by subjecting the viruses to energy-rich radiation, such as UV light or xcex3-rays. If desired, after treatment the pH can be adjusted to a value of about 7.
A vaccine containing the inactivated IBDV mutant can, for example comprise one or more of the above-mentioned pharmaceutically acceptable carriers or diluents suited for this purpose.
Preferably, an inactivated vaccine according to the invention comprises one or more compounds with adjuvant activity. Suitable compounds or compositions for this purpose include aluminium hydroxide, -phosphate or -oxide, oil-in-water or water-in-oil emulsion based on, for example a mineral oil, such as Bayol F(copyright) or Marcol 52(copyright) or a vegetable oil such as vitamin E acetate, and saponins.
The vaccine according to the invention comprises an effective dosage of the IBDV mutant as the active component, i.e. an amount of immunising IBDV material that will induce immunity in the vaccinated birds against challenge by a virulent virus. Immunity is defined herein as the induction of a significant higher level of protection in a population of birds after vaccination compared to an unvaccinated group.
Typically, the live vaccine according to the invention can be administered in a dose of 102-109 TCID50 infectious dose50 (TCID50) per animal, preferably in a dose ranging from 105.0-107.0 TCID50, and an inactivated vaccines may contain the antigenic equivalent of 105-109 TCID50 per animal.
Inactivated vaccines are usually administered parenterally, e.g. intramuscularly or subcutaneously.
Although, the IBDV vaccine according to the present invention may be used effectively in chickens, also other poultry such as turkeys, guinea fowl and partridges may be successfully vaccinated with the vaccine. Chickens include broilers, reproduction stock and laying stock.
The age of the animals receiving a live or inactivated vaccine according to the invention is the same as that of the animals receiving the conventional live- or inactivated IBDV vaccines. For example, broilers (free of maternally derived antibodies-MDA) may be vaccinated at one-day-old, whereas broilers with high levels of MDA are preferably vaccinated at 2-3 weeks of age. Laying stock or reproduction stock with low levels of MDA may be vaccinated at 1-10 days of age followed by booster vaccinations with inactivated vaccine on 6-8 and 16-20 weeks of age.
The invention also includes combination vaccines comprising, in addition to the IBDV mutant described above, one or more immunogens derived from other pathogens infectious to poultry or fish, respectively.
Preferably, the combination vaccine additionally comprises one or more vaccine strains of infectious bronchitis virus (IBV), Newcastle disease virus (NDV), egg drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV) or reovirus.